- 1 - Cell types Monocytes THP-1 cells Macrophages. LPS Treatment time (Hour) IL-6 level (pg/ml)
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1 Supplementary Table ST1: The dynamic effect of LPS on IL-6 production in monocytes and THP-1 cells after GdA treatment. Monocytes, THP-1 cells and macrophages (5x10 5 ) were incubated with 10 μg/ml of GdA for 48 hours. IL-6 secretion to the culture medium was measured by ELISA (N=8). IL-6 secretion to the culture medium of monocytes and THP-1 cells were measured after LPS activation for different duration (0-6 hours). Data are mean ± SEM. *P<0.05 when compared to corresponding control without GdA treatment. Cell types Monocytes THP-1 cells Macrophages LPS Treatment time (Hour) IL-6 level (pg/ml) ± ± ± ± ± ± ±275.9 GdA (10 μg/ml) ± ± ± ±652.9* ± ± ±185.9* ±719.2* - 1 -
2 Supplementary Table ST2: Effect of GdA on the phagocytic activity of monocytes/macrophages. Monocytes, macrophages and THP-1 cells (2x10 5 ) were incubated in 100 μl of culture medium containing 10 μg/ml GdA in a 96-well plate for 24 hours (N=9). Ten µl of latex beads coated with FITC-labeled rabbit-igg solution was then added. After 24 hours of incubation, trypan blue solution was added to quench the extracellular fluorescence. The intracellular fluorescent intensity of each well was measured with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The results are expressed as percentage of control without GdA treatment. Relative phagocytic activity = (Fluorescence of GdA-treated cells Fluorescence of blank)/(fluorescence of control cells Fluorescence of blank) x 100%. Data are mean ± SEM. *P<0.05 when compared to corresponding control without GdA treatment. Relative Phagocytic activity (%) GdA Monocytes ± 5.44 THP-1 cells ± 8.97 Macrophages ±
3 Supplementary Figure S1: Analysis of purified GdA in 12% SDS-PAGE with silver staining, western blot and the mass spectrometric analysis. (A) Purified GdA (0.5 μg/ml) was analyzed by silver staining (Lane 1) and western blotting (Lane 2). (B) Identification of GdA by MALDI-TOF-MS/MS of the ~26 kda protein band in Lane 1. The peptide sequence was obtained and searched against Homo sapiens proteins from NCBInr database. The protein sequence had total sequence coverage of 46%. A B Protein Accession no. Protein Peptide Sequence m/z Name Score Glycodelin IPI VHITSLLPTPEDNLEIVLHR DTTTPIQSMMCQYLAR VLVEDDEIMQGFIR AFRPLPR HLWYLLDLK QMEEPCRF
4 Supplementary Figure S2: Purification of blood monocytes and T-helper cell from female peripheral blood. Isolated monocyte and T-helper cells (5x10 5 ) were incubated with FITC-conjugated anti-human CD14 or FITC-conjugated anti-human CD4/APC-conjugated anti-human CD3 antibodies respectively, for 30 minutes on ice followed by the flow cytometric analysis. Isolated monocyte Isolated T-helper cell CD4-FITC CD14-FITC CD3-APC - 4 -
5 Supplementary Figure S3: Effect of GdA on the viability and cell death of monocytes/macrophages. (A) Cell viability was determined by XTT assay on monocytes, macrophages and THP-1 cells (3x10 4 ) after GdA treatment (0.01, 0.1, 1 and 10 μg/ml) for 72 hours (N=5). Freshly prepared XTT labeling mixture (50 μl) was added to the culture 6 hours before the end of the experiment. Data are expressed as percentage of control without GdA treatment = (Absorbance of GdA-treated cells Absorbance of blank) / (Absorbance of control cells Absorbance of blank) x 100%. (B) Viable, necrotic and apoptotic monocytes, THP-1 cells and macrophages (5x10 5 ) after GdA treatment (10 μg/ml) for 72 hours were quantified by bivariate Yo-Pro -1/PI flow cytometry (N=5). Cells negative for both Yo-Pro -1 and PI staining were counted as viable cells. Cells labeled with Yo-Pro -1 only were apoptotic cells, while those with Yo-Pro -1 and propidium iodide were necrotic cells. Data are mean ± SEM. A GdA (μg/ml) Viability (% of control) Monocytes THP-1 cells Macrophages ± ± ± ± ± ± ± ± ± ± ± ± 3.0 B Monocytes Blood monocyte Necrosis: 2.1 ± 1.0% Viable: 91.6 ± 2.5% Apoptosis: 6.3 ± 2.5% GdA (10μg/ml) Necrosis: 1.7 ± 0.9% Viable: 92.8 ± 2.3% Apoptosis: 5.6 ± 2.5% THP-1 cells THP-1 cell Necrosis: 5.3 ± 1.0% Viable: 89.3 ± 1.3% Apoptosis: 5.4 ± 3.1% GdA (10μg/ml) Necrosis: 6.4 ± 1,8% Viable: 87.7 ± 3.2% Apoptosis: 6.0 ± 1.5% GM-CSF-differentiated macrophage Macrophages Necrosis: 7.7 ± 5.3% Viable: 81.8 ± 2.6% Apoptosis: 10.6 ± 3.6% GdA (10μg/ml) Necrosis: 7.6 ± 3.4% Viable: 84.5 ± 1.4% Apoptosis: 7.9 ± 3.9% - 5 -
6 Supplementary Figure S4: Effects of ERK kinase, NF-κB and p38 inhibitors on viability of THP-1 cells. Cell viability was determined by the XTT proliferation assay on THP-1 cells incubated with ERK kinase inhibitors (PD98059: 10 μm; U0126: 1 μm), NF-κB inhibitors (CAPE and BAY-11708: 10 μm) or p38 inhibitors (of SB202190: 5 μm; SB203580: 10 μm) for 48 hours (N=12). Data are expressed as percentage of control without inhibitor treatment. Data are mean ± SEM. Stimulation Index (%) = (Absorbance of inhibitor treated cells Absorbance of blank) / (Absorbance of control cells Absorbance of blank) x 100%
7 Supplementary Figure S5: Expression of L-selectin in monocytes/macrophages. Monocytes, macrophages and THP-1 cells (5x10 5 ) were treated with anti-l-selectin-fitc followed by flow cytometry analysis (N=4)
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